The present invention relates to an apparatus and a method for the formation of frozen reagent beads, and more particularly, to an apparatus and method for the freeze forming of such beads comprised of a reagent used to carry out the analysis of a biological agent.
In the analysis of certain biological samples, it is common to use dry beads of specific reagents of a small precisely measured quantity. Such dried beads are commonly used in the analysis of blood assays but are also used with at variety of other analyses for other biological fluids and the reagent composition of the beads can also widely vary depending upon the particular analysis to be performed. As such, therefore, there is a need to produce dry reagent beads of a variety of compositions that are of a uniform size and predetermined characteristics by an apparatus or by using a method that is reliable and that can produce a large quantity of such beads at a rapid rate.
One of the methods of creating such dry chemical reagent beads is to use a freezing technique where the liquid reagent, in the desired amount and composition, is progressively dropped into a liquid cryogen, such as liquid nitrogen, where the liquid drop is fairly rapidly frozen into a reagent bead in a spherical configuration of the predetermined precise quantity. The frozen beads are thereafter harvested in a batch process and further processed by being lyophilized to form the ultimate product that is a dry bead of the desired reagent that dissolves quickly when in contact with the sample to be analyzed.
Thus, it is important to be able to rapidly create a large quantity on a continual basis of the frozen reagent beads such that the overall formation of such spherical beads, can be carried out efficiently and rapidly to produce the reagent beads of a constant, known quantity and contents.
In the production of the reagent bead, therefore, the liquid reagent is produced in the desired final concentration of constituents in liquid form and that liquid is dispensed in the form of drops that fall downwardly by means of gravity into a quantity of the liquid nitrogen. The liquid drop is thus frozen and eventually the frozen droplets fall to the bottom of the liquid nitrogen container. As the one drop is dispensed from the apparatus into the liquid nitrogen, the apparatus readies another drop that thus follows the prior drop after a predetermined elapsed time. The accumulated frozen beads of the particular reagent composition collect in the bottom of the container holding the liquid nitrogen and are periodically harvested, by a batch process, from the bottom of the container holding the liquid nitrogen and the process continued.
There is, therefore, a need for the aforesaid process to produce a large quantity of the final reagent beads in as rapid a period of time as is practical. At the present, the overall freezing process poses a problem to increasing the speed of the overall operation in that there is a physical limitation on the current process relating to the freezing process itself. As the spherical liquid drop is dispensed into the liquid nitrogen, the temperature of the liquid drop is relatively hot as compared with the temperature of the liquid nitrogen and thus, the initial contact between the liquid drop and the liquid nitrogen causes the liquid nitrogen to boil violently and generate a large quantity of nitrogen gas under the drop as it rests upon the surface of the liquid nitrogen.
In effect, the liquid drop floats upon the gaseous nitrogen and is supported so as to not be directly in contact with the much colder liquid nitrogen. That boundary layer of nitrogen gas isolates the liquid nitrogen from direct contact with the liquid drop of reagent and that boundary layer of the nitrogen gas is a poor thermal conductor, thus having a deleterious effect on the rate of freezing of the reagent drop. Obviously, in attempting to increase the rapidity of the overall freezing process, any parameter or effect that reduces the freezing rate is disadvantageous to the overall aim of the apparatus.
Accordingly, the freezing process itself is delayed by the boundary layer that impedes the rapid cooling and freezing of the drop of liquid reagent and thus imposes a severe limitation on the overall throughput of product since any subsequent drop cannot be dispensed until the prior drop has frozen and dropped to the bottom of the liquid nitrogen, otherwise, two drops may fuse together and create an extra large or double sized bead of the reagent and can introduce an inaccuracy with the use of that oversized reagent bead in carrying out a later analysis. However, due to the formation of the boiled nitrogen gas, the drop floats on the surface of the liquid cryogenic until it finally freezes and sinks to the bottom of the cryogen. The time for such freezing and dropping can vary and is dependent upon the size and density of the drop, however, a typical time can be in the order of about six seconds.
Therefore, there is a timewise constraint on the overall freezing process, and left alone, would pose a serious hindrance to any effort at speeding up the present freezing process that depends highly upon the freezing rate of the liquid drop of reagent. It should be noted that the formation of periodic double drops cannot fully and successfully be alleviated through the use of passing all of the frozen reagent beads through a sieve material to try to capture the larger, oversized beads as the particular orientation of the beads as they pass through such a sieve can render the use of a sieve unreliable and thus still not fully solve the problem, that is, even a double bead can, at times, be in the proper orientation so as to pass through a sieve that is sized for a single bead. In addition it is believed to be a better course of action to solve the problem in the first place rather than resort to a remedial effort to minimize the problem of the formation of double sized beads, and thus, a solution to the initial problem would be preferable.
One currently known apparatus for the formation of the frozen reagent beads is shown and described in U.S. Pat. No. 5,275,016 of Chatterjee et al, where an apparatus is provided that has a rotating carousel on which is situated a plurality of trays containing the cryogenic liquid. In the Chatterjee et al patent, therefore, the carousel is rotated so as to position the trays beneath a liquid dispensing means where the drops of reagent are deposited in the liquid filled trays and the carousel continuously rotates. The difficulty with a rotating carousel, however, is that the throughput is also limited by the physical dimensions of the overall apparatus, thus, as one attempts to increase the throughput to achieve a higher production rate of frozen drops of reagent, the diameter of the rotating carousel has to increase outwardly at a drastic rate and cause the overall apparatus to become exceedingly large in order to produce any appreciable increase in the rate of production. This is an inefficient use of the space available to the user with the employment of a rotating carousel that creates a limitation on the rate of production of the frozen reagent drops of the Chatterjee et al apparatus. Too, with a rotary carousel, the linear speed of the carousel varies depending upon the radial position of the drops that fall into the carousel.
In addition, in the Chatterjee et al apparatus, it is stressed that the continuous rotational movement of the carousel, as opposed to a stop and go or intermittent movement, is intended to create a smooth movement so as to prevent agitation of the cryogenic liquid. However, as has been previously discussed, there is a layer of nitrogen gas that forms directly under the liquid drop of reagent as it is in the freezing process resting on the surface of the liquid cryogen and which forms a boundary layer that impedes the freezing process. As such, and to the contrary of the Chatterjee et al smooth movement, it would be preferable to create some agitation or movement of the cryogenic liquid to disrupt that layer of nitrogen gas as such disruption will accelerate the freezing process and allow for a faster rate of production of the frozen beads of reagent.
A further apparatus for the production of frozen droplets is shown and described in U.S. Pat. No. 4,982,577 where the liquid cryogen, such as liquid nitrogen, is caused to flow downwardly along a sluiceway to eventually enter a reservoir, however the entire apparatus including the sluiceway is contained within an insulated vessel in an attempt to minimize the evaporation of the nitrogen which is otherwise very susceptible to such evaporation along the sluiceway and within the reservoir.
Accordingly, it would be advantageous to provide an apparatus and a method for forming frozen beads of a reagent in a manner such that the production rate can be sufficiently high so as to generate the needs of such frozen beads in an expeditious and efficient manner and with enhanced efficiency and use of the available space.
Now then, in accordance with the present invention, there is provided an apparatus and a method of producing a high rate of uniform frozen beads of a reagent for use in a chemical analysis. In the present invention, therefore, there is provided an apparatus that comprises a heavily insulated tray, preferably of stainless steel, and which contains a quantity of the liquid cryogen, such as liquid nitrogen. Within the tray is a removable grid that is rectangular in configuration and which is, in turn, divided into a plurality of individual cells that are preferably square in cross section. Each cell is made up of cell walls forming a open volume for the free flow of the cryogen through out all of the cells since each cell is open at its bottom so that the liquid cryogen can freely fill each cell from the bottom of the tray up to a uniform level or depth. In the preferred embodiment, the cells are aligned in a plurality of rows along an x axis and the rows are in abutting relationship along the y axis, with each cell providing an isolating environment from the adjacent cell or cells.
The apparatus further comprises a liquid dispenser that causes one drop of liquid reagent at a time to fall, by gravity, downwardly into the liquid nitrogen contained within the tray. There may, in order to optimize the process and increase its throughput, be multiple liquid drop dispensers and, in the preferred embodiment, two of such dispensers may be utilized. Each drop of the liquid reagent is made up of a material in a homogeneous form that is to be used in a later analysis.
The tray filled with liquid nitrogen is positioned upon an x-y positioning table that allows the table to move the tray in directions along both the x and the y axis of that tray. Such tables are commercially available and can be customized for the particular movement desired with respect to speed and movement direction. The typical movement of an x-y table is a stepwise or intermittent motion. In the operation of the present apparatus, therefore, a drop of the liquid reagent is allowed to fall from the liquid dispenser into a predetermined cell within the plurality of cells and the cell is located in that position by the translation of the x-y table and is at rest. After the drop has fallen to the surface of the liquid nitrogen and contained within that designated cell, the x-y table moves the tray along the x axis or y axis so that the translation brings an adjacent cell into alignment beneath the liquid dispenser so that a subsequent drop of a liquid reagent can be dispensed into that next cell. As the apparatus progresses, each drop is successively allowed to fall into subsequently located cells until, in the preferred embodiment, the entire row of cells positioned along the x axis has received a drop of liquid reagent and the x-y table then moves the tray in the direction of the y axis to allow a new row of cells to be utilized.
As such, therefore, the x-y table continues to translate the position of the tray containing the liquid nitrogen along that second row of cells until the end of that row is reached, whereupon the x-y table then moves the tray in the direction of the y axis again to progress to the next row. In such manner, the x-y table progresses through all of the rows of the isolating cells and, in each instance, the drop of liquid reagent is dispensed into an individual cell and the x-y table thus moves the tray to present the next cell into position to receive the next drop of liquid reagent. In the preferred embodiment, the pattern of stepwise movement can be established so as to advance the tray one cell at a time to return the tray, eventually, to its original position with the initial cell in position to receive the drop for a subsequent cycle.
In the aforedescribed manner, each drop is allowed to remain on the surface of the liquid nitrogen in an isolated cell where it freezes and ultimately drops to the bottom of the steel tray, free from the cells. Thus, the formation of a double drop is prevented as the isolation provided by the grid forming the cell walls of each cell prevents any two drops from coming in contact with each other during the freezing process. Once totally frozen, however, the problem of the drops sticking together to form a double drop is alleviated and the frozen drops can thus simply fall and accumulate in the bottom of the tray and be removed by batch process whenever a predetermined number of drops has been formed.
Accordingly, the overall cycle or positioning, stepwise, of all of the cells to receive a drop of liquid reagent can be established to consume sufficient time to assure that the freezing of the first drop in that cycle has completely frozen. Thus, the cells, or more specifically, the cell walls, can extend from just above the surface of the liquid nitrogen i.e. about 1 cm. above, to an intermediate depth below the surface of the nitrogen so that, once the drops are fully frozen into beads, all of the beads can sink to the bottom of the tray to amass together at the bottom where they can remain until removed from the apparatus. At the completion of an entire cycle, the grid can be removed from the tray, leaving the frozen beads at the bottom of the tray to be transferred to a lyophilizer.
Alternative means of harvesting the beads at the bottom of the tray of liquid nitrogen can be used, as one such means, the tray can be constructed so as to have a sloped or inclined bottom surface so that the frozen beads congregate at the lower end of the angled bottom and thus are easier to remove. A further alternate embodiment would be to include some conveyer system at the bottom surface of the tray to automatically remove the frozen beads.
In addition, with the present apparatus, the movement of the tray by means of the x-y table, is preferably an intermittent or stepwise motion, that is, the table positions the applicable cell beneath the liquid drop dispenser where the table is momentarily at rest. After the drop has been administered to the cell currently in the receiving position, the x-y table then moves the tray so as to align the next cell into that receiving position.
Thus, the movement or motion of the tray is not intended to be a smooth, even movement as used in the Chatterjee et al patent to prevent disruption of the liquid nitrogen. To the contrary, with the present apparatus, that intermittent motion causes a deliberate, controlled agitation to the liquid nitrogen to disrupt of the layer of gaseous nitrogen that forms underneath the liquid drop as it rests on the surface of the liquid nitrogen and therefore positively enhances the freezing of the drop to form the frozen bead. Since any disruption of the layer of gaseous nitrogen improves the freezing process, that intermittent motion, itself, increases the throughput of the overall apparatus by speeding the freezing process of the liquid drop of reagent. It should be noted that the controlled agitation is not a jerking motion that would severely disrupt the movement of the tray, but is a controlled acceleration and deceleration such as to cause ripples on the surface of the cryogen and that acceleration and deceleration may be symmetrical or asymmetrical.
In addition, with typical x-y tables for the positioning and translation of the cells of the present invention, the acceleration profile of the table motion can be controlled by the normal software provided with such commercially available x-y tables so as to increase or decrease that agitation to suit the desires of the user by changing the acceleration and deceleration to produce the optimal effect of the freezing process.
These and other characteristics of the present invention will become apparent through reference to the following detailed description of the preferred embodiments and accompanying drawings.